Bioinformatics Analysis, Expression Profiling, and Functional Characterization of Heat Shock Proteins in Wolfi-poria cocos.

Heat shock proteins (HSPs) play critical roles in regulating different mechanisms under high-temperature conditions. HSPs have been identified and well-studied in different plants. However, there is a lack of information about their genomic organization and roles in medicinal plants and fungi, especially in Wolfi-poria cocos (W. cocos). We identified sixteen heat shock proteins (HSPs) in W. cocos and analyzed in terms of phylogenetic analysis, gene structure, motif distribution patterns, physiochemical properties, and expression comparison in different strains. Based on phylogenetic analysis, HSPs were divided into five subgroups (WcHSP100, WcHSP90, WcHSP70, WcHSP60, and WcsHSP). Subgroups WcHSP100s, WcHSP90s, WcHSP70s, WcHSP60, and WcsHSPs were further divided into 3, 2, 3, 1, and 6 subfamilies, respectively. Moreover, the expression profiling of all HSP genes in five strains of W. cocos under different temperature extremes revealed that expression of most HSPs were induced by high temperature. However, every subfamily showed different expression suggesting distinctive role in heat stress tolerance. WcHSP70-4, WcHSP90-1, and WcHSP100-1 showed the highest response to high temperature stress. Heterologous expression of WcHSP70-4, WcHSP90-1, and WcHSP100-1 genes in Escherichia coli enhanced survival rate of E. coli during heat stress. These findings suggest the role of W. cocos heat shock genes in the high temperature stress tolerance.

Screening of novel HSP-inducing compounds to conserve cardiomyocyte function in experimental atrial fibrillation.

BACKGROUND: The heat shock protein (HSP) inducer, geranylgeranylacetone (GGA), was previously found to protect against atrial fibrillation (AF) remodeling in experimental model systems. Clinical application of GGA in AF is limited, due to low systemic concentrations owing to the hydrophobic character of GGA. OBJECTIVES: To identify novel HSP-inducing compounds, with improved physicochemical properties, that prevent contractile dysfunction in experimental model systems for AF. METHODS: Eighty-one GGA-derivatives were synthesized and explored for their HSP-inducing properties by assessment of HSP expression in HL-1 cardiomyocytes pretreated with or without a mild heat shock (HS), followed by incubation with 10 µM GGA or GGA-derivative. Subsequently, the most potent HSP-inducers were tested for preservation of calcium transient (CaT) amplitudes or heart wall contraction in pretreated tachypaced HL-1 cardiomyocytes (with or without HSPB1 siRNA) and Drosophilas, respectively. Finally, CaT recovery in tachypaced HL-1 cardiomyocytes posttreated with GGA or protective GGA-derivatives was determined. RESULTS: Thirty GGA-derivatives significantly induced HSPA1A expression after HS, and seven showed exceeding HSPA1A expression compared to GGA. GGA and nine GGA-derivatives protected significantly from tachypacing (TP)-induced CaT loss, which was abrogated by HSPB1 suppression. GGA and four potent GGA-derivatives protected against heart wall dysfunction after TP compared to non-paced control Drosophilas. Of these compounds, GGA and three GGA-derivatives induced a significant restoration from CaT loss after TP of HL-1 cardiomyocytes. CONCLUSION: We identified novel GGA-derivatives with improved physicochemical properties compared to GGA. GGA-derivatives, particularly GGA*-59, boost HSP expression resulting in prevention and restoration from TP-induced remodeling, substantiating their role as novel therapeutics in clinical AF.

Euphornin reduces proliferation of human cervical adenocarcinoma HeLa cells through induction of apoptosis and G2/M cell cycle arrest.

BACKGROUND: The plant Euphorbia helioscopia L. has been used in traditional Chinese medicine for treating various disorders such as tuberculosis and edema. The aim of this study was to investigate the effect of euphornin, a bioactive compound isolated from E. helioscopia, on proliferation of human cervical adenocarcinoma HeLa cells by analyzing cell viability, rate of apoptosis, and cell cycle progression. MATERIALS AND METHODS: The sulforhodamine B assay was used to study the effect of euphornin on the proliferation of HeLa cells. Morphological changes to cell nuclei were identified after Hoechst 33342 staining. Mitochondrial membrane depolarization (MMP) was analyzed after staining with JC-1 dye. The influence of euphornin on the apoptosis rate was analyzed by Annexin V/propidium iodide double staining. Fluorescence-activated cell sorting was applied to investigate the influence of euphornin on cell cycle progression. Proteins were obtained from HeLa cells and analyzed by Western blots. RESULTS: A cell viability assay showed that euphornin inhibited proliferation of HeLa cells in a dose-dependent and time-dependent manner. Euphornin also induced apoptosis in a concentration-dependent manner, with the rates of apoptosis ranging from 25.3% to 52.6%. A high concentration of euphornin was found to block HeLa cells at the G2/M stage. A Western blot analysis suggested that euphornin might exhibit antitumor activity by inducing apoptosis. Euphornin treatment altered the ratio of Bax/Bcl-2 in HeLa cells, which led to the release of cytochrome complex. The levels of cleaved caspase-3, caspase-8, caspase-9, and caspase-10 were also markedly increased by euphornin treatment. Analysis of cell cycles indicated that euphornin induced cell cycle arrest by increasing the level of the phospho-CDK1 (Tyr15) protein. The various assays demonstrated that euphornin treatment resulted in a significant suppression of cell growth accompanied by G2/M cell cycle arrest and increased rate of apoptosis via mitochondrial and caspase pathways. CONCLUSION: Our findings suggest that euphornin has the potential to be used as a cancer therapeutic agent against human cervical adenocarcinoma.

[Progress on molecular biology of Isaria farinosa, pathogen of host of Ophiocordyceps sinensis during the artificial culture].

Isaria farinosa is the pathogen of the host of Ophiocordyceps sinensis. The present research has analyzed the progress on the molecular biology according to the bibliometrics, the sequences (including the gene sequences) of I. farinosa in the NCBI. The results indicated that different country had published different number of the papers, and had landed different kinds and different number of the sequences (including the gene sequences). China had published the most number of the papers, and had landed the most number of the sequences (including the gene sequences). America had landed the most numbers of the function genes. The main content about the pathogen study was focus on the biological controlling. The main content about the molecular study concentrated on the phylogenies classification. In recent years some protease genes and chitinase genes had been researched. With the increase of the effect on the healthy of O. sinensis, and the whole sequence and more and more pharmacological activities of I. farinosa being made known to the public, the study on the molecular biology of the I. farinosa would be deeper and wider.

Review on Natural Enemies and Diseases in the Artificial Cultivation of Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes).

Ophiocordyceps sinensis (syn. Cordyceps sinensis), well known as DongChongXiaCao (DCXC), is one of the most valuable traditional Chinese medicinal species. In this article, we provide a systematic review of natural enemies and diseases encountered in artificial cultivation of DCXC. Unfortunately, DCXC has been endangered over the past decades due to overharvesting and a worsening ecological environment. Therefore, the artificial cultivation of DCXC has been extensively investigated in recent years. Complete indoor artificial cultivation and semi-field cultivation are the two most common strategies used to cultivate DCXC. However, cultured DCXCs are often attacked by various natural enemies and diseases, which have resulted in substantial loss of the valuable medicinal resource. In this study, we have summarized the species of natural enemies and types of diseases confronted by DCXC. Twenty reported natural enemy species are categorized into four classes, one of which is reported for the first time in this study. Moreover, six microbial pathogens are also discussed. The recapitulation of the natural enemies and diseases in DCXC artificial cultivation not only promote the development of integrated pest management of DCXC cultivation but also provide important information to help preserve and develop this valuable resource.

[Effect of wound to growth of larva of host to Ophiocordyceps sinensis during artificial breeding].

To clear the effect of the wound to the growth of the larva of the host to the Ophiocordyceps sinensis, the wounds of same severity at the same position were made artificially to the larva and which were artificial fed at the same environment and condition. The results indicated that, over the winter, the survival rate of the wounded of the infection larva was lower than that of the healthy larva, but the weight had no significant difference between the wounded and the healthy larva. The survival rate of the wounded of the no infection larva was lower than that of the healthy larva, but except with black skin, the wounded larva with offwhite and dusty red had no influence on the variety of the weight. In summery, wound had no advantage to the survival rate, but had no influence to the weight. The result had provided theoretical basis to the reforming of the system of the artificial culture O. sinensis.

[Studies on the chemical constituents from the leave of Paulownia tomentosa].

OBJECTIVE: To study the chemical constituents of the leave of Paulownia tomentosa. METHODS: The constituents were isolated by column chromatography and their structures were elucidated through spectroscopic analysis. RESULTS: The compounds were identified as apigenin(I), uteolin(II), homoeriodictyol(III), 3alpha-hydroxyurs-12-en-28-oic acid (IV),3beta,19alpha-dihydroxyurs-12en-28-oic acid (pomolic acid)(V),2alpha,3a-dihydroxyurs-12-en-28-oic acid(VI),ursolic acid (VII),2alpha,3beta-dihydroxyolean-12-en-28-oic acid (maslinic acid) (VIII), daucosterol(IX),beta-sitosterol(X). CONCLUSION: The compound I-X are obtained from the leave of Paulownia tomentosa (Thunb.) Steud for the first time.

[Cloning and expression of novel swine gene BCL-G(L) in E.coli and preparation of its polyclonal antibody in guinea pigs].

AIM: In order to express a novel gene named as BCL-G(L); of swine in E.coli and prepare its polyclonal antibody. METHODS: The contig sequence of the gene was predicted and in silicon cloned by blasting the human BCL-G(L); in swine ESTs database in NCBI. The cloning sequence was obtained by RT-PCR from swine spleen. The cloning sequence was identified by sequencing and compared with the contig sequence. Then the gene was cloned into a prokaryotic expression vector pET-32a to construct a recombinant plasmid named as pET32a-BCL-G(L);. The fusion protein pET32a-BCL-G(L); was expressed in E.coli BL21 and purified using a His-tag fusion protein purification kit. Then guinea pigs were immunized with the purified protein to get the specific polyclonal antibody. RESULTS: The titer of the antibody was 1:800 detected by ELISA. The protein BCL-G(L); can be specifically detected by western blot assay using the polyclonal antibody. CONCLUSION: The novel swine gene BCL-G(L); was cloned and expressed in E.coli and its polyclonal antibody was prepared successfully.

[Studies on the chemical constituents from flower of Paulownia fortunei].

OBJECTIVE: To study the chemical constituents of the flower of Paulownia fortunei. METHODS: The constituents were isolated by column chromatography and their structures were elucidated through spectroscopic analysis. RESULTS: The compounds were identified as: apigenin (I), luteolin (II), Hesperetin (III), Naringenin-7-O-beta-D-glucoside (IV), Arbutin (V), 4-hydroxybenzyl-beta-D-glucoside (VI), abscisic acid (VII) and 1-acetoxy-3-hydroxypropan-2-yl-3-hydroxypentanoate (VIII). CONCLUSION: All these compounds are obtained from the flower of this plant for the first time.

[Study on biological character of Hepialus introduced from Yunnan province].

OBJECTIVE: To study the biological character of Hepialus introduced from Yunnan province. METHOD: H. jianchuanensis introduced from Yunnan was bred artificially to observe its biological character. RESULT: The pupa duration was about 40 d, pupa emergence lasted for one day, female to male ratio was about 1.2:1, the mating peak was 19:30-20:30, the adults duration was about 25 d, the average life time of female was 5-6 d and that of male 6-7 d, the average number of the producing egg was about 300, the average weight of the 100 eggs was 10.33 mg. Lasting period of the larva is 350 d and the infancy larva showed the character of the polyphagy and cold endurance. CONCLUSION: Results indicate that there are some differences in the whole duration, weight of the 100 eggs and the number of the eggs left in the abdomen between H. jianchuanensis and others bred at the Kangding base.

[Effects of slow release-fertilizers on yield, nutrient contents and quality of Coptis chinensis].

OBJECTIVE: To study the effects of four kind of slow-release fertilizers on yield and quality of Coptis chinensis. METHOD: One to three years C. chinensis was fertilized with slow-release fertilizers twice in April and in September. The yield and nutrient content along with quality of C. chinensis were measured after two years growth. RESULT AND CONCLUSION: All of the slow-release fertilizers increased the yield obviously, and the effect of SRF1 and SRF4 is the most significant. Comparing with control group, the N content in aerial part of 1-2 year-old C. chinensis treated with SRF1 and SRF4 was lower and P and K were higher than that of control group, and the N content in aerial part of 3 year-old C. chinensis treated with SRF1 and SRF4 was higher and P and K were higher than that of control group; The N content in the root of land 3 year-old C. chinensis treated with SRF1 and SRF4 showed no significant difference comparing with control group, and P and K were lower than that of control group, the N and P content in root of 2 year-old C. chinensis treated with SRF1 and SRF4 was higher and K were lower than that of control group. After two years growth berberine content of C. chinensis treated with SRF1, SRF2 and SRF3 were significantly increased, and total alkaloid content of C. chinensis treated with SRF1, SRF3 and SRF4 were significantly increased. We recommend that SRF4 is used as the special fertilizer for 1-year-old C. chinensis, and the SRF1 and SRF4 for 2-year-old C. chinensis, and the SRF1 for 3-year-old C. chinensis.

[Effects of integrated pest control techniques to growth of host larvae Cordyceps sinensis].

UNLABELLED: To study the effects of the integrated pest control techniques on growth of host larvae of Cordyceps sinensis. METHOD: The integrated pest control techniques were compared with conventional techniques to evaluate the effects on growth of host larvae. RESULT AND CONCLUSION: The results showed that the techniques had broken the balance of the microbial living in the material, produced effective inhibition on the pests, raised the survival rate and promoted the growth of the host larvae at the same time.